Procedure Development for Identification of Histamine from Chicken Meat

doi:10.1038/protex.2013.099

Method Article

Procedure Development for Identification of Histamine from Chicken Meat

https://doi.org/10.1038/protex.2013.099

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In the present protocol we describe a novel TLC procedure that can sufficiently reduce the time required to isolate Histamine from biological sample like chicken meat (10g, dried basis), 96 minutes. Furthermore, Histamine isolation on TLC was best achieved when chicken meat sample was stirred at full speed (1600 RPM) on heated magnetic stirrer plate. This helps in liberating tissues bound histamine using a low temperature range, 80C0. Beyond this temperature however, viscosity of the chicken meat occurs that makes trouble in filtration of the sample. Whatman filter paper (No.1) is sufficient to filter the samples in 7.0 minutes. However, vacuum filtration or squeezing the sample contained filter paper may further reduce the time. Using TLC running buffer, methyl alcohol: ammonia (20:1) is not sufficient to rapidly mobilize TLC spots upward, instead methyl alcohol: ammonia (30:1.5) is reasonable to ablate time. TLC method is sensitive in this case and can detect histamine even other biogenic amines of biological importance, food etc. Overall, TLC procedure is satisfactory, sensitive, rapid, and above all reproducible; therefore, TLC is a good option than expensive protocol like HPLC method.

Biological techniques

Histamine

chicken meat

food safety

TLC

Biogenic amines (e.g. histamine) are nitrogenous and low molecular weight organic compounds found in food products. Fermented food products and meat are greatly exposed to this threat due enzymes assisted decarboxylation of free amino acids (1). Several microbes have been implicated in production of allergenic substance like histamine. United State and other countries have put strict regulations for biogenic amines in foods due to the fact histamine and others biogenic amines are potentially toxic to humans when their level cross acceptable level. Presently, Histamine content should be limited to 200 mg/kg, and level above 1000 mg/kg are toxic, The Rapid Alert System for Food and Feed, RASFF (2). Higher level of biogenic amines indicates excessive food fermentation or poor food storage wherein enzyme decarboxylase positive microorganisms implicated. Hence, biogenic compounds give clue regarding spoilage of food commodities. Meat and food products with high content of proteins thus serve as basis for rapid spoilage.

1-Methyl Alcohol (Sigma Aldrich, USA)

2-Ammonia (Sigma Aldrich, USA)

3-Ninhydrine (Sigma Aldrich, USA)

(1) TLC plate (TLC silica Gel 60 F25, size 10x20; DC kieselgel 60 F254, Merck KGA, 64271 Germany)

(2) Hot Plate and Magnetic Stirrer (Model MS-300)

(3) Oven (China Series No 0378, Type DL 102A)

(4) Micro-pipette

(5) Spectroline (R) MiniMAX (TM) Portable UV Hobbyist's Lamp

Chicken meat sample (dried powder) ten grams were added to 150 mL methyl alcohol in pyrex flask and placed in oven at 70 Centigrade for 7 minutes. CRITICAL we suggest 5-7 minutes placement of the sample in oven, longer placement of sample in oven not only evaporate methy alcohol but also burn sample.
Sample containing flask was placed on hot plate magnetic stirrer and was rapidly stirred (1600 RPM) at 80 centigrade for 8 minutes. CRITICAL increasing temperature beyond the level and increasing the time lead to a more viscous sample formation which ultimately makes problem in filtration.
Stirred sample was filter using whatman NO1 filter paper and this takes 7 minutes time. CRITICAL squeezing once or twice the sample decreased filtration time or alternatively, vacuum filtration may be good.
The filter extract was taken in micropipette, 0.5 microliter and was spotted on TLC plate along with standard histamine (0.5 microliter) and this procedure was completed in one hour. CRITICAL 30mL methyl alcohol and 1.5mL ammonia is sufficient to smoothly mobilize the analyte/sample spot.
2% ninhydrin solution was made (w/v, acetone). This takes only 3 minutes.
TLC plate was dried in air and then sprayed with ninhydrin.
The TLC plate was air dried, and visualized using Spectroline (UV long Wave). Then TLC plate was photographed. This step takes 10 minutes.

sample preparation and placing sample in oven takes 8 minutes.
stirring of the sample also takes 8 minutes.
Sample filtration takes 7 minutes.
Ninhydrin solution prepartion takes 3 minutes
Spotting sample on TLC and running TLC takes 60 minutes
Photographing spot under UV takes 10 minutes to complete procedures.

Taking completely dried samples and putting it into 50 mL methy alcohol make the solution dried during oven drying or when put on magnetic stirer. Hence, 150 mL methyl alcohol overcome on this trouble. similarly, putting samples for longer time in oven (more than 10 minutes) make viscous the sample. hence, 8 minutes is sufficient for this step.
Increasing Magnetic hot plate temperature greater than 80 centigrade again leading to a viscous solution even a very dried sample. Also, exceeding this temperature range repel the content of the sample that stick the walls of the flask.
Filtration though filter paper although provide easy way but filtration aided by vacuum pumping would further made process satisfactory.
Using 20mL methyl Alcohol and 1mL ammonia is not sufficient for TLC mobile phase. using this ratio greatly increased time and hence; 30:1.5 is preferable.

The present protocols is developed for the rapid identification of biogenic amines using TLC as a cost effective tool.

Silla Santos, M. H. Biogenic amines: Their importance in foods. Int. J. Food Microbiol. 29, 213-231 (1996).
Sawilska-Rautenstrauch D, Gawarska H, Karlowski K. [Analysis of notifications on exceeding of the acceptable level of histamine according to the RASFF reports]. Rocz Panstw Zakl Hig. 62, (4):365-369 (2011).
Lin JS, Baranowski JD, Olcott HS. Rapid thin-layer chromatographic-densitometric determination of histamine in tuna. J Chromatogr. 130 426-430 (1977).

We are grateful to the department of Food Science & Human Nutrition, University of Veterinary and Animal Sciences, Lahore-Pakistan for providing research facility and space.

The authors declare no competing financial interest

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Procedure Development for Identification of Histamine from Chicken Meat

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Abstract

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Procedure Development for Identification of Histamine from Chicken Meat

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Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Acknowledgements

Additional Declarations

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Version 1

Privacy Policy

Terms of Service

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